(1) Technical Field
The present invention relates to a medium, apparatus and method for separating a cell group, or a cell population or subpopulation of interest from a suspension of animal cells, in particular adhesive animal cells.
(2) Prior Art and Its Problems:
Separating a cell group of interest, i.e. a desired cell group type, or cell population or subpopulation, from a cell suspension is a routine practice conducted in the medical field, not only for the purpose of clinical testing but also with a view of performing immunodiagnosis or immunotherapy, including basic evaluation of a series of biophylactic potentiators. However, if one wants to separate T cells, B cells, K cells or NK cells from a suspension of lymphocytes, there is no reliable method available for doing so. Reliable, reproducible methods are unavailable for isolating a certain cell population while retaining the isolated cells in their original, that is, unchanged state. A strong need, therefor, exists for providing a technique which is capable of separating a cell population of interest in its unchanged state from a cell suspension.
Japanese Patent Application (OPI) Nos. 204454/82 and 140886/81 (the term "OPI" as used herein means an "unexamined published Japanese patent application") proposed techniques which are useful for obtaining T cells by a single stage of separation procedure using particulate materials having acidic functional groups or hydrophobic and water-insoluble particulate materials having micropores, more particularly, homo- and copolymers of ethylene, propylene, vinyl chloride, vinyl acetate, styrene, divinylbenzene, acrylonitrils and methyl methacrylate, or nylon, polycarbonate, polyethylene terephthalate or polyester copolymers, or derivatives thereof, incorporating a sulfonic acid group, a carboxyl group, a phosphonic acid group or a phenol group. Problems experienced with these prior art cell separation techniques are that the column of particulate materials is easily plugged because of a small average particle size and that the chemical polymers per se or their residual low-molecular weight substances are toxic to the activity of the cells to be recovered.
While several materials and methods are authorized by international academic societies for use in separation of cells, all of them require considerable time and careful treatment in making preparation for separation procedures, involve complicated, time-consuming and laborious separating operations, and fail to provide a high degree of reproducibility because the resolution and its pattern (the spectrum of a cell population of interest) provided by the separating medium differs considerably from one production lot to another.
The Sephadex G10 process using a crosslinked dextran separation medium is one of the internationally approved methods for cell separation. Although the operating theory of this typical cell separating technique has not been completely unravelled, the principal role is believed to be played by the difference in adhesion to the dextran material between two cells such that macrophages or larger adhesive accessary cells are retarded by their relative adhesion whereas T cells or B cells do not adhere well to the dextran medium. However, in this method, non-adherent small accessary cells also do not adhere to the dextran medium. In addition, a recent study has revealed that some of the subpopulations of T cells are adherent on the dextran so that the T cell population cannot be obtained in its complete and integral form. This is a substantial drawback in attempt to use the Sephadex G10 process as an immunodiagnostic technique.
Another internationally approved cell separating device is a column packed with nylon wool. This column may be used as a means for obtaining a separated cell group abundant with T cells, but the substantial yield of the target cell that can be obtained by this column is generally low (12-25%). Although the purity of the T cell population obtained from this column is fairly high, the recovered T cell population differs from that in the original cell suspension. The column is used after being packed with a predetermined amount of nylon wool, but the resolution and its pattern will not only vary from one production lot to another but will also depend upon the manner in which the wool fibers are disintegrated, packed in the column or washed. It is to be noted that this problem of variations in characteristics is common to the organic separating mediums.
As will be understood from the foregoing brief explanation, commercial application of methods involving the separation of cells by making use of their adhesive nature has just begun. Higher process efficiency, speed and precision are expected to result from improving the separating method per se or the separating medium used, or from developing new materials suitable for use as separating mediums. Concerted efforts toward this goal are desired not only in the general field of biotechnology but also in important medical fields such as immunodiagnosis and immunotherapy.